Calcium content and distribution as a function of growth and transformation in the mouse 3T3 cell

Total Ca content and that fraction of Ca sensitive to removal by the chelator ethylene glycol-bis(β-aminoethyl ether)N,N,N',N'-tetraacetate (EGTA) have been investigated in the mouse 3T3 cell as a function of growth stage, transformation with SV40 virus, and serum levels of the media. Cells were allowed to grow through several doublings in media containing (45)Ca. The cellular content of (45)Ca was used to access total cell Ca. That fraction of (45)Ca removed by EGTA was presumed to represent primarily surface-localized Ca. The data are expressed on a per cell volume basis to compensate for size differences as a function of growth stage and transformation. During exponential growth phase, the 3T3 cell contains 525pmol Ca/μl cell volume. Of this, approx. 457 pmol/μl is not removable by EGTA and, presumably, is cytoplasmically located. This value is in close agreement with previous studies on the HeLa cell (470 pmol Ca/μl cell water after the removal of the surface Ca). The low level of EGTA- removable Ca present in the 3T3 cell during early exponential growth (68 pmol Ca/μl cell volume) increases progressively with increasing cell density, and upon quiescence it is sevenfold greater. In contrast, SV40- transformed 3T3 cells growing exponentially possess total levels of Ca which are approximately two-thirds the levels of the normal 3T3 cell. However, their EGTA-sensitive Ca is not significantly different from that of exponentially growing, normal 3T3 cells. As the transformed cells continue to grow at high density, their total ca and their sensitivity to EGTA do not change, in contrast to the normal 3T3 cell. Thus, an increase in Ca associated with the cell surface appears to be correlated with growth inhibition. This has been investigated further by regulating growth of the normal and transformed cell with alterations in the serum level of the media. In 4 percent calf serum the normal cell is stopped from continued proliferation. Growth stoppage under these conditions is characterized by a nearly fourfold increase in EGTA-removable Ca, similar to the increase observed upon quiescence in depleted 10 percent serum. Similar treatment of the transformed cell does not reduce its growth rate, nor does it significantly alter Ca distribution. However, at 0.5 percent medium serum levels, the SV40 3T3 growth rate is substantially reduced and, under these conditions, EGTA-removable Ca increases twofold.     

Recombinant adeno-associated virus type 2-mediated gene delivery into the <Emphasis Type="Italic">Rpe65</Emphasis><Superscript>-/-</Superscript> knockout mouse eye results in limited rescue

Background  

Leber's congenital amaurosis (LCA) is a severe form of retinal dystrophy. Mutations in the RPE65 gene, which is abundantly expressed in retinal pigment epithelial (RPE) cells, account for approximately 10–15% of LCA cases. In this study we used the high turnover, and rapid breeding and maturation time of the Rpe65 ^-/- knockout mice to assess the efficacy of using rAAV-mediated gene therapy to replace the disrupted RPE65 gene. The potential for rAAV-mediated gene treatment of LCA was then analyzed by determining the pattern of RPE65 expression, the physiological and histological effects that it produced, and any improvement in visual function.     

Natal homing in juvenile loggerhead turtles (Caretta caretta)

Juvenile loggerhead turtles (Caretta caretta) from West Atlantic nesting beaches occupy oceanic (pelagic) habitats in the eastern Atlantic and Mediterranean, whereas larger juvenile turtles occupy shallow (neritic) habitats along the continental coastline of North America. Hence the switch from oceanic to neritic stage can involve a trans-oceanic migration. Several researchers have suggested that at the end of the oceanic phase, juveniles are homing to feeding habitats in the vicinity of their natal rookery. To test the hypothesis of juvenile homing behaviour, we surveyed 10 juvenile feeding zones across the eastern USA with mitochondrial DNA control region sequences (N = 1437) and compared these samples to potential source (nesting) populations in the Atlantic Ocean and Mediterranean Sea (N = 465). The results indicated a shallow, but significant, population structure of neritic juveniles (PhiST = 0.0088, P = 0.016), and haplotype frequency differences were significantly correlated between coastal feeding populations and adjacent nesting populations (Mantel test R2 = 0.52, P = 0.001). Mixed stock analyses (using a Bayesian algorithm) indicated that juveniles occurred at elevated frequency in the vicinity of their natal rookery. Hence, all lines of evidence supported the hypothesis of juvenile homing in loggerhead turtles. While not as precise as the homing of breeding adults, this behaviour nonetheless places juvenile turtles in the vicinity of their natal nesting colonies. Some of the coastal hazards that affect declining nesting populations may also affect the next generation of turtles feeding in nearby habitats.     

Reproductive Biology of the Smooth Dogfish, Mustelus canis, in the Northwest Atlantic Ocean

Shark populations tend to be highly vulnerable to overexploitation due to low fecundity and slow growth rates. Recent fishing pressure on the smooth dogfish in the northwest Atlantic has created a necessity for more information about their basic biology. Length and age at maturity, fecundity, and reproductive season were estimated. Total length at 50% maturity was 102cm for females, and 86cm for males. The majority of females were mature at age 4 or 5, and all males were mature at 2 or 3 years of age. Females had an 11–12 month gestation with parturition occurring in May, ovulation occurring between May and July likely in late May and early June, and mating occurring between May and September. Fecundity ranged between 3 and 18 pups per litter, and was positively related to length and age, with a mean of 9.53 pups per litter. Sperm was found in the terminal zone of the oviducal gland of females throughout the year.     

The Finland-United States investigation of non-insulin-dependent diabetes mellitus genetics (FUSION) study. I. An autosomal genome scan for genes that predispose to type 2 diabetes

We performed a genome scan at an average resolution of 8 cM in 719 Finnish sib pairs with type 2 diabetes. Our strongest results are for chromosome 20, where we observe a weighted maximum LOD score (MLS) of 2.15 at map position 69.5 cM from pter and secondary weighted LOD-score peaks of 2.04 at 56.5 cM and 1.99 at 17.5 cM. Our next largest MLS is for chromosome 11 (MLS = 1.75 at 84.0 cM), followed by chromosomes 2 (MLS = 0.87 at 5.5 cM), 10 (MLS = 0.77 at 75.0 cM), and 6 (MLS = 0.61 at 112.5 cM), all under an additive model. When we condition on chromosome 2 at 8.5 cM, the MLS for chromosome 20 increases to 5.50 at 69.0 cM (P=.0014). An ordered-subsets analysis based on families with high or low diabetes-related quantitative traits yielded results that support the possible existence of disease-predisposing genes on chromosomes 6 and 10. Genomewide linkage-disequilibrium analysis using microsatellite marker data revealed strong evidence of association for D22S423 (P=.00007). Further analyses are being carried out to confirm and to refine the location of these putative diabetes-predisposing genes.     

Ontogenetic changes in the diet of the sandbar shark, <Emphasis Type="Italic">Carcharhinus plumbeus</Emphasis>, in lower Chesapeake Bay and Virginia (USA) coastal waters

This study describes the diet of the sandbar shark, Carcharhinus plumbeus, highlighting differences in diet within various regions of the Virginia (USA) nursery area, as well as ontogenetic changes in diet. Stomach samples were obtained in 2001 and 2002 from 232 sharks caught by gillnets or longlines. Historical data from the Virginia Institute of Marine Science (VIMS) Shark Ecology Program were also analyzed. Ontogenetic changes in diet were evident, with crustacean prey decreasing in frequency with increasing shark size, and elasmobranch prey importance increasing with increasing shark size. Whereas previous research in Chincoteague Bay, VA showed the blue crab, Callinectes sapidus, was the dominant crustacean in sandbar shark diet, the mantis shrimp, Squilla empusa, dominated the crustacean portion of the diet in this study. Differences in diet of sharks were observed among locations within the study area. Small juveniles (≤80 cm precaudal length) in the lower Chesapeake Bay ate more fishes, whereas Eastern Shore juveniles ate more crustaceans. Crustacean prey items varied among locations along the Eastern Shore, with more portunid crabs consumed in waters near Wachapreague and more mantis shrimp consumed near Sand Shoal Inlet. Our study showed that Carcharhinus plumbeus is a generalist predator and is thus unlikely to strongly impact the population of any particular prey species, and in turn is not likely to be strongly affected by fluctuations in abundance of a single prey species.     

Global conservation priorities for marine turtles

Where conservation resources are limited and conservation targets are diverse, robust yet flexible priority-setting frameworks are vital. Priority-setting is especially important for geographically widespread species with distinct populations subject to multiple threats that operate on different spatial and temporal scales. Marine turtles are widely distributed and exhibit intra-specific variations in population sizes and trends, as well as reproduction and morphology. However, current global extinction risk assessment frameworks do not assess conservation status of spatially and biologically distinct marine turtle Regional Management Units (RMUs), and thus do not capture variations in population trends, impacts of threats, or necessary conservation actions across individual populations. To address this issue, we developed a new assessment framework that allowed us to evaluate, compare and organize marine turtle RMUs according to status and threats criteria. Because conservation priorities can vary widely (i.e. from avoiding imminent extinction to maintaining long-term monitoring efforts) we developed a “conservation priorities portfolio” system using categories of paired risk and threats scores for all RMUs (n = 58). We performed these assessments and rankings globally, by species, by ocean basin, and by recognized geopolitical bodies to identify patterns in risk, threats, and data gaps at different scales. This process resulted in characterization of risk and threats to all marine turtle RMUs, including identification of the world''s 11 most endangered marine turtle RMUs based on highest risk and threats scores. This system also highlighted important gaps in available information that is crucial for accurate conservation assessments. Overall, this priority-setting framework can provide guidance for research and conservation priorities at multiple relevant scales, and should serve as a model for conservation status assessments and priority-setting for widespread, long-lived taxa.     

Age and growth of the sandbar shark, Carcharhinus plumbeus, in Hawaiian waters through vertebral analysis

Age and growth estimates were determined for the sandbar shark, Carcharhinus plumbeus, from Oahu, Hawaii in the central Pacific Ocean. Age estimates were obtained through vertebral centra analysis of 187 sharks. We verified our age estimates through marginal increment analysis of centra and oxytetracycline marking methods of at liberty sandbar sharks. Sizes of sampled sharks ranged from 46 to 147 cm pre-caudal length. Four growth models were fitted to length-at-age data; two forms of the von Bertalanffy growth model, the Gompertz growth model, and a logistic growth model. Males and females exhibited statistically significant differences in growth, indicating that females grow slower and attain larger sizes than males. Growth parameter estimates revealed slower growth rates than previously estimated (based on captive specimens) for Hawaiian sandbar sharks. The von Bertalanffy growth model using empirical length-at-birth provided the best biological and statistical fit to the data. This model gave parameter estimates of L _∞ = 138.5 cm PCL and k = 0.12 year⁻¹ for males and L _∞ = 152.8 cm PCL, k = 0.10 year⁻¹ for females. Male and female sandbar sharks mature at approximately 8 and 10 years of age, respectively.